Transport vehicles for macromolecules

ABSTRACT

The invention relates to new compounds with the general formula I for use as a tool to introduce macromolecules into cells. The invention further relates to compositions for introducing macromolecules into cells, comprising vesicles formed by at least one compound in a solvent.The macromolecule can be incorporated in the vesicles and/or bound to the vesicles or another aggregate of the new compounds. In a preferred embodiment at least one targeting molecule, for instance a (labelled) antibody, may further be attached to the vesicles

CROSS REFERENCE TO RELATED APPLICATION

This is a division of application Ser. No. 08/686,045, filed Jul. 24,1996, now U.S. Pat. No. 5,853,694.

BACKGROUND OF THE INVENTION

The present invention related to new compounds which are capable ofintroducing macromolecules into eucaryotic cells.

The introduction of macromolecules, including DNA, proteins and thelike, into eucaryotic cells can be carried out in different ways, forinstance by means of transport vehicles. Such vehicles introduce amolecule into the cell, for instance by means of endocytosis. Thevehicles may bind, but for instance also encapsulate, the molecules tobe transported. In the latter case the vehicles are referred to asvesicles. Known vesicles are liposomes which consist of a bilayer ofphospholipids.

Liposomes are for instance used to introduce medicines into the cell. Itappeared that liposomes are incorporated into the cell both in vivo andin vitro by means of endocytosis (Nandi, P. K. et al. (1986) J. Biol.Chem. 261:16722; Heath, T. D. (1987) Methods Enzymol. 149:111). Thismeans that the largest portion of the material which is incorporated inthe cell will ultimately appear in the lyposomal apparatus, where itwill be decomposed. Particularly for substances which have their effectin the cytoplasm or the nucleus this is obviously very disadvantageous.

If the substances to be introduced are hydrophilic it will be difficultto introduce them into liposomes. The main portion of the materialremains in the aqueous phase. Particularly in case of expensivesubstances, like probes and many medicines, this is an obviousdisadvantage.

To prevent that the substances to be introduced into the cell end up inthe cell by means of endocytosis, attempts have been made to usefusogenic phospholipids as transport vehicles. When fusogenicphospholipids have bound (hydrophilic) substances and have formedvesicles, the vesicles will introduce there substances into the cellafter fusion with the cell membrane. However, such attempts have notproven to be very successful because fusogenic liposomes have a strongtendency to mutually merge instead of fusing with the cell membrane(Fonteijn, T. A. A., Ph.D. Thesis (1992)).

One of the most important applications in which molecules are introducedinto a cell is transfection of the (eucaryotic) cell with DNA or RNA.Transfection is being used for studying the function and regulation ofgenes and proteins, but also for the genetic modification ofmicro-organisms, plants and animals. There is a large number ofartificial techniques which allow DNA to be introduced into a cell,including DNA-micro-injection, DNA-coprecipitation within inorganicsalts or with polycations, DNA-encapsulation in liposomes, and makingthe cell membrane permeable with the aid of chemical or physical means.

A more recent technique involves the use of cationic amphiphilicmolecules as transport vehicles. One of the best-known amphiphiles isthe quaternairy ammonium amphiphile DOTMA(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride) whichin combination with dioleyol phosphatidyl ethanolamine (DOPE), iscommercially available with the name Lipofectinem™. Both molecules arelipidic(analogues), which form liposomes, which will form complexes withthe negatively charged nucleic acids. Supposedly, the liposomes mergewith the plasma membrane and introduce in this way nucleic acids intothe cell. However, it could also be done by means of endocytosis. Theexact mechanism is yet unknown. With the aid of Lipofectinem™ thetransfection efficiency may be enhanced by a factor of 30 with respectto other known systems, including the classical calcium phosphateprecipitation method. However, the disadvantage of Lipofectinel™ is itstoxicity and therefore it may be difficult or not possible to use it invivo. Therefore, a demand still remains for other and bettertransfection methods.

SUMMARY OF THE INVENTION

It is the aim of the present invention to provide new cationicamphiphilic compounds, which allow high efficiencies, for theintroduction into a cell of nucleic acids and other macromolecules,including for example proteins and medicines.

The aim of the invention is achieved by compounds of general formula I:

in which:

R₁ is a

(a) branched or linear (C₁-C₅)alkyl, or

(b) branched or linear ((C₁-C₅)alkyl)aryl, or

(c) branched or linear ((C₁-C₅)alkyl)N⁺(CH₃)₃, or

(d) branched or linear ((C₁-C₅)alkyl)₂R₂,R₃,R₄-pyridinium in compliancewith formula I

X⁻ is a halide counter ion chosen from Cl⁻, I⁻ or Br, and in which

R₃ is hydrogen and R₂ and R₄ are identical or different and are selectedfrom the group consisting of branched or linear: (C₁₀-C₂₀)alkyl, mono-or polyunsaturated (C₁₀-C₂₀)alkenyl, (C═O)—O—(C₁₀-C₂₀)alkyl,O—(C═O)—(C₁₀-C₂₀)alkyl, or ((C₁₀-C₂₀)alkyl)aryl, or

R₂ and R₄ are hydrogen and R₃ is CHR₅R′₅ in which R₅ and R′5 areidentical or different and are selected from the group consisting ofhydrogen branched or linear: (C₁₀-C₂₀)alkyl, mono- or polyunsaturated(C₁₀-C₂₀)alkenyl, (C═O)—O—(C₁₀-C₂₀)alkyl, O—(C═O)—(C₁₀-C₂₀)alkyl, or((C₁₀-C₂₀)alkyl)aryl.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the relative efficiencies of transfection ofCOS-7 cells using the synthetic amphiphile SAINT-2 or SAINT-2/DOPE incomparison to transfection efficiency with DOTMA/DOPE.

FIG. 2 is a graph showing the relative efficiencies of transfection ofBHK cells using the synthetic amphiphile SAINT-2/DOPE or DOTMA/DOPE.

FIG. 3 is a graph showing the relative efficiencies of transfection ofcells using the synthetic amphiphiles DOTMA/DOPE, SAINT-2, andSAINT-2/DOPE in comparison to transfection efficiency with calciumphosphate precipitation.

DETAILED DESCRIPTION OF THE INVENTION

A particularly advantageouis compound according to the invention is1-methyl-4-(19-cis,cis-hepatritiaconta-9,28-dienyl) pyridinium chloride(SAINT-2). The compounds according to the invention are all based on apyridine ring, which is at one or two positions substituted by a long(ar)alkyl chain. It has been found that with the amphiphiles accordingto the invention, and particularly with the compound here referred to as“SAINT-2”, a transfection efficiency can be obtained which, dependent onthe cell type, is at least eight times higher as that of Lipofectine™.

With SAINT-2/DOPE it also proved to be possible to introduce proteins,particularly gelonine (30 kD), into the cell. Other cell types,particularly Baby Hamster Kidney (BHK) cells, may be transfected. Thisis impossible with Lypofectine™ for BHK cells. SAINT-2/DOPE yields evenbetter results with BHK cells than Lipofectine™ with COS-7 cells.

The compounds according to the invention may be synthesized in awell-known fashion. The synthesis will be further illustrated in theexamples.

The amphiphiles according to the invention may be used in a large numberof applications.

The transport into the cell of nucleic acids and their derivatives is ofimportance for transfection. The aim of transfection is, for instance,to make proteins or to perform research. Furthermore, transfectednucleic acids, possibly labelled with streptavidine or radioactivelylabelled, may be used for in situ hybridisation. A more advancedapplication is to influence gene expression, for instance blocking ofgenes by antisense strands. Furthermore, gene expression may also bestimulated. Furthermore, the defect genes may be replaced. The lattertwo applications are of particular importance in gene therapy.

The advantage of compounds according to the invention is that they, ascompared to the known transport vehicles, can be used in much lower,non-toxic concentrations. Probably, they also do not cause animmunologic response.

If DNA and/or RNA are to be introduced into a cell the compounds and thenucleic acids have to be mixed in a certain ratio. It has been foundthat for the known amphiphiles, including DOTMA, there exists an optimumamphiphile concentration (Felgner, P. L. et al. (1987) Proc. Natl. Acad.Sci. USA 84:7413). The transfection efficiency again reduces if acertain amount is exceeded. A comparable situation also holds for thecompounds according to the invention.

The cationic amphiphiles according to the invention may also be used totransport negatively charged proteins, including gelonine in particular,into the cell.

The amphiphiles may also be used to transport substances likecytostatics. Lipophilic cytostatics in particular do interact with thecompounds according to the invention and may in this way be introducedinto the cell very efficiently.

In a preferred embodiment of the invention the transport vehicles may bepurposely brought to a specific site by mixing the amphiphiles with atargeting molecule, such as, for instance, an antibody which is directedagainst an epitope in the neighbourhood of the site where theincorporated substance has to excercise its activity. The antibody ispreferably coupled to the amphiphilic compound but it may also becoupled, for instance, through a spacer, to the substance to betransported.

The antibody may be labeled such as with a radioactive label or astreptavidine label. In order to facilitate the translocations of DNA orother macromolecules across the cell membrane the compounds according tothe invention may also be mixed with a phospholipid or with each other.

The present invention will be illustrated in further detail with bymeans of the accompanying examples which are only serve as anillustration and do not limit the scope of the invention.

EXAMPLES Example 1 Synthesis

Compounds with the general structure formula

may be divided in a number of groups dependent on their substituents.The synthesis of four of those groups will be given below as an example.

1. 4-Substituted N-Alkylpyridinium Salts

1.1. Synthesis of 1-Methyl-4-(1-octadecylnonadecyl)pyridinium Chloride.

The compound is synthesised according to scheme 1 below as described byE. J. R. Sudhölter in his Ph.D. thesis at the University of Groningen,1981, page 37.

1.2. Synthesis of1-Methyl-4-(19-cis,cis-heptatritiaconta-9,28-dienyl)pyridinium Chloride(SAINT-2).

Scheme 2 describes the sequence of the reactions.

The synthesis has been carried out under nitrogen. 2.226 g (0.022 mol)of di-isopropyl amine was dissolved in 15 ml of dry diethyl ether. Then13.8 ml (1.6 M) n-butyl lithium in n-hexane was added dropwise at 0° C.Subsequently, the mixture was stirred for 10 minutes. This mixture wasadded dropwise to 0.931 g (0.01 mol) 4-picoline in 10 ml of diethylether at −20° C. After this it was stirred for another 30 minutes. Thecolour of the reaction mixture became deeply orange. Then 7.567 g (0.020mol) oleyl iodide (85% cis) in 5 ml of diethyl ether was added oneportion. The temperature increased to 0° C. while stirring.Subsequently, the mixture was stirred during one night at roomtemperature. The next day 100 ml of diethyl ether was added to thereaction mixture and subsequently 40 ml of H₂O. The organic layer wasseparated and washed with 3 portions of 30 ml H₂O. The ether layer wasdried on Na₂SO₄, filtered and condensed. The residu (5.9 g) is a viscousbrown oil which was purified over a column of 100 g neutral Al₂O₃ (act.2-3). As eluent a mixture of n-hexane-diethyl ether (8:2) was used. 4.32g (0.0073 mol) 4-(19-cis,cis-heptatritiaconta-9,28-dienyl)pyridine wasobtained (intermediate 1b2, yield 73%).

NMR data: ¹H NMR(CDCl₃): δ 0.89 (t, 6H); 1.27 (chain, 52H); 2.0 (m, 8H);2.43 (tr, 1H); 5.34 (m, 4H); 7.06 (d, J_(H,H)=6 Hz, 2H); 8.49 (d,J_(H,H)=6 Hz, 2H). ¹³C NMR: δ 14.0 (CH₃); 22.6; 27.1; 27.3; 29.1; 29.2;29.4; 29.5; 29.6; 29.7; 31.8; 36.1 (CH₂-chain); 45.5 (CH); 123.1 (CH)129.7 (CH); 129.8 (CH); 149.5 (CH); 155.3 (C).

1.527 g (0.0025 mol) of intermediate 1 was dissolved in 10 ml ofacetone. Subsequently, 2 ml of methyl iodide was added and the mixturewas boiled for 3 hours. After evaporation of the solvent a light yellowbrown viscous oil was obtained with a yield of 0.8 g (intermediate 1b1,yield 97%).

NMR data: ¹H NMR(CDCl₃): δ 0.85 (t, 6H); 1.23 (chain, 44H); 1.55 (m,4H); 1.73 (m, 4H); 2.00 (m, 8H); 2.77 (m, 1H); 4.7 (2, 3H); 5.31 (m,4H); 7.74 (d, J_(H,H)=6.7 Hz, 2H); 9.31 (d, J_(H,H)=6.7 Hz, 2H). ¹³CNMR: δ 13.9 (CH₃); 22.4; 26.9; 27.2; 28.9; 29.1; 29.3; 29.4; 29.5; 31.6;35.4 (CH₂-chain); 46.4 (CH); 48.3 (N—CH₃); 126.8 (CH); 129.5 (CH); 129.7(CH); 144.9 (CH); 167.1 (C).

0.4 g (0.00054 mol) of intermediate 2 was dissolved in 3 ml of methanoland this solution was eluted with methanol over a Dowex column (1*8,200-400 mesh Cl⁻ form). The compound 1b was obtained as a viscous oil ina yield of 0.319 g (0.00049 mol 92%).

NMR data: ¹H NMR(CDCl₃): δ 0.87 (t, 6H); 1.26 (CH₂-chain, 44H); 1.57 (m,4H); 1.75 (m, 4H); 2.00 (m, 8H); 2.77 (m, 1H); 4.77 (s, 3H); 5.32 (m,4H); 7.15 (d, J_(H,H)=6.2 Hz, 2H); 9.50 (d, J_(H,H)=6.2 Hz, 2H).

1.3. Synthesis of 1-(1-Butyl-N,N,N-trimethylAmmonium)-4-(17-tritiacontanyl)pyridinium Chloride.

This compound was synthesized according to scheme 3 below.

2. 3,5-Disubstituted-N-alkylpyridinium Salts

The general synthesis according to scheme 4 below was described in theliterature by Sudhölter (vide supra) and Wang et al., J. Org. Chem. 42,1286 (1977).

2.1. 1-Methyl-3,5-dicarbo-N-octadecyloxy)pyridinium Chloride.

This compound was synthesized according to scheme 4.

NMR data: ¹H NMR(CDCl₃): δ 0.85 (t, 6H); 1.30 (chain, 64H); 4.40 (t,4H); 5.03 (s, 3H); 9.20 (t, 1H); 10.00 (d, 2H).

3. 4-Substituted-N-alkyl Pyridinium Salts

The synthesis was described by F. J. A. Hundscheid and J. B. F. N.Engberts, J. Org. Chem. 49, 3088 (1984).

3.1. 1-Methyl-4((-n-hexadecyloxy)carbonyl)pyridinium Iodide.

The synthesis of this compound and its characterisation are described byHundscheid and Engberts (vide supra).

NMR data: ¹H NMR(CDCl₃): δ 0.9 (t, 3H); 1.25 (m, 28H); 4.35 (t, 2H);4.70 (s, 3H); 8.35 (d, 2H); 9.35 (d, 2H).

4. 4-Substituted-N-aralkyl Pyridinium Salts

4.1. 1-(3-Phenyl-1-propyl)-4-n-dodecylpyridinium Iodide.

The compound was synthesized by boiling a mixture of 2.26 g (9.2 mmol)1-iodo-3-phenyl propane and 2.57 g (10.0 mmol) 4-n-dodecylpyridine in 35ml of dry acetone for 16 hours. The solvent was evaporated and theyellow solid substance was recrystallized from THF/ether. The yield is3.06 g (6.2 mmol), melting point 79.0-80.0° C.

NMR data: ¹H NMR(CDCl₃): δ 0.83 (t, 3H); 1.21 (chain, 20H); 1.61 (m,2H); 2.36 (m, 2H); 2.78 (m, 2H); 4.89 (t, 2H); 7.05-7.20 (m, 5H); 7.73(d, 2H); 9.30 (d, 2H).

Example 2 Formation of Unilamellar Vesicles

A suitable amount of lipid was dried under N₂(g). In case ofcombinations of substances these are first mixed and then dried. Thelipid film layer is subsequently dried further under vacuum. The lipidsare then suspended, vortexed and subsequently sonicated in a suitablevolume of water until the solution is clear.

Example 3 Transfection of Eucaryotic Cells by Compounds According to theInvention

DNA and unilamellar vesicles, as prepared in Example 2, are both broughtinto Hepes buffered saline (HBS, pH 7.4; both 0.5 ml) and subsequentlymixed. The DNA/amphiphile complex is directly formed. In a typicaltransfection experiment 1 μg of DNA and 7.5-10 μg of the amphiphileSAINT-2 (1-methyl-4-(19-cis,cis-heptatritia contadienyl-9-28) pyridiniumchloride) or 1 μg of DNA and 10-15 μg of total amphiphile (SAINT-2/DOPE1:1) is used.

Cells in six-well plates, which are cofluent by 70-80%, are washed twicewith 1 ml of HBS and subsequently 1 ml of the DNA/amphiphile complex wasadded per well. The cells were incubated during 4 hours at 37° C. afterwhich 1 ml Dulbecco Modified Eagle Medium (DMEM) supplemented with 10%Foetal Calf Serum (FCS) was added. After an incubation of 16 hours at37° C. the medium was exchanged by 2 ml fresh DMEM with 10% FCS. After asubsequent incubation of 28 hours at 37° C. the cells were gathered. Thecells were washed twice with a phosphate buffered saline (PBS) andscraped in 300 μl 1× lysis buffer (Promega). The scraped cells wereincubated for 10 minutes at 56° C. and subsequently centrifuged atmaximum speed for two minutes at room temperature. On the supernatant anenzyme determination (CAT-assay) and a protein determination (Lowry)were carried out.

100 μl of the cell extract was incubated together with 3 μl¹⁴C-chloramphenicol (25 mCi/l), 5 μl N-butyryl-CoA (2 mg/ml) and 17 μl0.25 M Tris.HCL (pH 8.0) during 90 minutes at 37° C. The reaction wasstopped by adding 0.3 ml of mixed xylenes (Aldrich). The samples werevortexed for 30 seconds and subsequently centrifuged at maximum speedfor 3 minutes at room temperature. The organic phase was again extractedwith 0.1 ml 0.25 M Tris.HCL, vortexed for 30 seconds and centrifuged for3 minutes. 4 ml of counting fluid was added to 0.2 ml of the organicphase and the radio activity was measured.

It was found that transfection of COS-7 cells with the new amphiphile(SAINT-2 and SAINT/DOPE) is eight times more efficient that that withDOTMA/DOPE vesicles (see FIG. 1).

It appeared that with the new amphiphile also other cell types,including for instance BHK cells, can be transfected (FIG. 2). When astable transfection is carried out with the new amphiphile it appearedto be possible to transfect 42-45% of the COS-7 cells. With DOTMA-DOPEvesicles on average 25-29% of the cells are transfected.

Example 4 Transport of Proteins in an Eucaryotic Cell

The synthetic amphiphile SAINT-2 is, in combination with DOPE, asuitable agent for the delivery of proteins into cells. The efficiencyof protein internalisation with SAINT-2/DOPE as a carrier can bemonitored with the aid of the gelonine protein. Internalized geloninespecifically inhibits the protein synthesis of cells and thisinhibitation is a direct measure for the amount of gelonine which hasbeen brought into the cell. Unilamellar versicles of the synthetic agentamphiphile SAINT-2 and DOPE are obtained by bath sonication. Gelonine isadded to a certain concentration (0-20μM) SAINT-2/DOPE in HBS from astock solution (2 mg/ml).

CV-1 cells, grown in twelve-well plates, are washed three times withHBS. Subsequently, the cells are incubated for 1 hour at 37° C. with theamphiphile/gelonine complex in HBS obtained in this way. After this thecells are again washed three times with HBS.

The inhibition of protein synthesis by gelonine is being followed bydetermining the building-in of radioactively labelled methionine intothe treated cells. This is carried out by incubating the cells for 30minutes with 1 μCi³⁵S-methionine. Subsequently, the cells are washedthree times with PBS and finally scraped in 10% TCA. The cell lysateobtained in this way is washed three times with 10% TCA and the amountof radioactive methionine present in the cell lysate is determined withthe aid of a scintillation counter.

Incubation of CV-1 cells with the amphiphile/gelonine complex gives astrong inhibition of the protein synthesis with respect to the controlexperiment in which the cells were incubated with the syntheticamphiphile only. At a concentration of 5 μM SAINT-2/DOPE and 1.6 μMgelonine an inhibition of protein synthesis of 50% was obtained.

Example 5 Toxicity Studies

To determine the toxicity of the compound SAINT-2 according to theinvention with respect to DOTMA-DOPE the COS-7 cells are incubated withdifferent concentrations of both lipid samples. The residual proteincontent is taken as a measure for the amount of surviving cells.

A decrease of the protein content from 2 to 1 mg/ml was observed forDOTMA-DOPE starting from 71 μM lipid. For SAINT-2 a decrease from 2 to1.75 mg/ml was found starting from 90 μM.

This shows that SAINT-2 is clearly less toxic than DOTMA-DOPE.

We claim:
 1. Compounds with the general formula I

in which: R₁ is a (a) branched or linear (C₁-C₅)alkyl, or (b) branchedor linear ((C₁-C₅)alkyl)aryl, or (c) branched or linear((C₁-C₅)alkyl)N⁺(CH₃)₃, or (d) branched or linear((C₁-C₅)alkyl)₂R₂,R₃,R₄-pyridinium in compliance with formula I X⁻ is ahalide counter ion chosen from Cl⁻, I⁻ or Br⁻, and in which R₃ ishydrogen and R₂ and R₄ are identical or different and are selected fromthe group consisting of branched or linear: (C₁₀-C₂₀)alkyl, mono- orpolyunsaturated (C₁₀-C₂₀)alkenyl, (C═O)—O—(C₁₀-C₂₀)alkyl,O—(C═O)—(C₁₀-C₂₀)alkyl, and ((C₁₀-C₂₀)alkyl)aryl, or R₂ and R₄ arehydrogen and R₃ is CHR₅R′₅ in which R₅ and R′₅ are identical ordifferent and are selected from the group consisting of branched orlinear: (C₁₀-C₂₀)alkyl, mono- or polyunsaturated (C₁₀-C₂₀)alkenyl,(C═O)—O—(C₁₀-C₂₀)alkyl, O—(C═O)—(C₁₀-C₂₀)alkyl, and((C₁₀-C₂₀)alkyl)aryl, wherein disclaimed are the compounds with thegeneral formula I in which R₁ is CH₃, R₂ and R₄ are hydrogen, R₃ is(C₁₆H₃₃)₂CH and X⁻ is all mentioned counter ions (Cl⁻, I⁻, Br⁻) anddisclaimed are the compounds in which R₁ is CH₃, R₂ and R₄ areC₁₆H₃₃—O—(C═O), R₃ is hydrogen and X⁻ is all mentioned counter ions(Cl⁻, I⁻, Br⁻).
 2. Compounds according to claim 1, characterized in thatR₁ is CH₃, R₂ and R₄ are hydrogen, R₃ is (C₁₈H₃₇)₂CH and X is Cl⁻, Br⁻,I⁻.
 3. Compounds according to claim 1, characterized in that R₁ is(CH₂)₄—N⁺(CH₃)₃, R₂ and R₄ are hydrogen, R₃ is (C₁₈H₃₅)₂CH and X is Cl⁻,Br⁻, I⁻.
 4. Compounds according to claim 1, characterized in that R₁ isCH₃, R₂ and R₄ are C₁₈H_(“)—O—C(O), R₃ is hydrogen and X is Cl⁻, Br⁻,I⁻.
 5. Compounds according to claim 1, characterized in that R₁ is CH₃,R₃ is hydrogen, R₂ and R₄ are (C═O)—O—(C₁₀-C₂₀)alkyl, mono- orpolyunsaturated (C═O)—O—(C₁₀-C₂₀)alkenyl or(C═O)—O—((C₁₀-C₂₀)alkyl)aryl, and X⁻ is Cl⁻, Br⁻, or I⁻.
 6. Compoundsaccording to claim 1, characterized in that R₁ is CH₃, R₃ is hydrogen,R₂ and R₄ are not identical and each represents a group with the formula(C═O)—O—(C₁₀-C₂₀)alkyl, mono- or polyunsaturated(C═O)—O—(C₁₀-C₂₀)alkenyl, or (C═O)—O—((C₁₀-C₂₀)alkyl)aryl, and X⁻ isCl⁻, Br⁻, or I⁻.
 7. Compounds according to claim 1, characterized inthat R₁ is CH₃, R₂ and R₄ are hydrogen, R₃=CHR₅R′₅, and in which R₅ andR′₅ are identical or different and are selected from the groupconsisting of (C═O)—O—(C₁₀-C₂₀)alkyl, mono- or polyunsaturated(C═O)—O—(C₁₀-C₂₀)alkenyl, or (C═O)—O—((C₁₀-C₂₀)alkyl)aryl, and X⁻ isCl⁻, Br⁻, or I⁻.
 8. Compounds according to claim 7, characterized inthat R₅ and R′₅ are (C═O)—O—C₁₆H₃₃.
 9. Compounds according to claim 1,characterized in that R₁ is (CH₂)_(n)—C₆H₅, in which n=3-6, R₂ and R₄are hydrogen, R₃=CHR₅R′₅, R₅ and R′₅ are identical or different and areselected from the group consisting of branched or linear: (C₁₀-C₂₀)alkyland mono- or polyunsaturated (C₁₀-C₂₀)alkenyl; and X⁻ is Cl⁻, Br⁻ or I⁻.10. Compounds according to claim 9, characterized in that R₁ is(CH₂)₃—C₆H₅, R₂ and R₄ are hydrogen, R₅ and R′₅ are n-C₁₂H₂₅ and X⁻ isCl⁻, Br⁻ or I⁻.
 11. Compounds according to claim 1, characterized inthat R₁ is ((CH₂)₄)R₂,R₃,R₄-pyridinium, in which R₂ and R₄ are hydrogen,R₃ is (C₁₈H₃₅)₂CH, and X⁻ is Cl⁻, Br⁻, or I⁻.
 12. A composition tointroduce macromolecules into cells, comprising a mixture of a compoundaccording to claim 1 and at least one macromolecule.
 13. The compositionaccording to claim 12, further comprising at least one targetingmolecule.
 14. The composition according to claim 13, wherein thetargeting molecule is an antibody.
 15. The composition according toclaim 14, wherein the antibody is radioactively labeled or labeled withstreptavidine.
 16. The composition according to claim 12, wherein themacromolecule is a nucleic acid.
 17. A method of introducingmacromolecules into cells comprising: a. forming a mixture comprising acompound according to claim 1 and at least one macromolecule; and b.contacting cells with said mixture.